Molecular Recognition in C-Type Lectins: The Cases of DC-SIGN, Langerin, MGL, and L-Sectin.

Carbohydrates play a pivotal role in intercellular communication processes. In particular, glycan antigens are key for sustaining homeostasis, helping leukocytes to distinguish damaged tissues and invading pathogens from healthy tissues. From a structural perspective, this cross-talk is fairly complex, and multiple membrane proteins guide these recognition processes, including lectins and Toll-like receptors. Since the beginning of this century, lectins have become potential targets for therapeutics for controlling and/or avoiding the progression of pathologies derived from an incorrect immune outcome, including infectious processes, cancer, or autoimmune diseases. Therefore, a detailed knowledge of these receptors is mandatory for the development of specific treatments. In this review, we summarize the current knowledge about four key C-type lectins whose importance has been steadily growing in recent years, focusing in particular on how glycan recognition takes place at the molecular level, but also looking at recent progresses in the quest for therapeutics.

Glycosylated Cell-Penetrating Peptides (GCPPs).

The cell membrane regulates the exchange of molecules and information with the external environment. However, this control barrier hinders the delivery of exogenous bioactive molecules that can be applied to correct cellular malfunctions. Therefore, the traffic of macromolecules across the cell membrane represents a great challenge for the development of the next generation of therapies and diagnostic methods. Cell-penetrating peptides are short peptide sequences capable of delivering a broad range of biomacromolecules across the cellular membrane. However, penetrating peptides still suffer from limitations, mainly related to their lack of specificity and potential toxicity. Glycosylation has emerged as a potential promising strategy for the biological improvement of synthetic materials. In this work we have developed a new convergent strategy for the synthesis of penetrating peptides functionalized with glycan residues by an oxime bond connection. The uptake efficiency and intracellular distribution of these glycopeptides have been systematically characterized by means of flow cytometry and confocal microscopy and in zebrafish animal models. The incorporation of these glycan residues into the peptide structure influenced the internalization efficiency and cellular toxicity of the resulting glycopeptide hybrids in the different cell lines tested. The results reported herein highlight the potential of the glycosylation of penetrating peptides to modulate their activity.

Unraveling Sugar Binding Modes to DC-SIGN by Employing Fluorinated Carbohydrates.

A fluorine nuclear magnetic resonance (19F-NMR)-based method is employed to assess the binding preferences and interaction details of a library of synthetic fluorinated monosaccharides towards dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN), a lectin of biomedical interest, which is involved in different viral infections, including HIV and Ebola, and is able to recognize a variety of self- and non-self-glycans. The strategy employed allows not only screening of a mixture of compounds, but also obtaining valuable information on the specific sugar-protein interactions. The analysis of the data demonstrates that monosaccharides Fuc, Man, Glc, and Gal are able to bind DC-SIGN, although with decreasing affinity. Moreover, a new binding mode between Man moieties and DC-SIGN, which might have biological implications, is also detected for the first time. The combination of the 19F with standard proton saturation transfer difference (1H-STD-NMR) data, assisted by molecular dynamics (MD) simulations, permits us to successfully define this new binding epitope, where Man coordinates a Ca2+ ion of the lectin carbohydrate recognition domain (CRD) through the axial OH-2 and equatorial OH-3 groups, thus mimicking the Fuc/DC-SIGN binding architecture.

Structure and N-acetylglucosamine binding of the distal domain of mouse adenovirus 2 fibre.

Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple ß-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutagenesis, we show that the fibre specifically binds to the monosaccharide N-acetylglucosamine (GlcNAc). The crystal structure of the complex reveals that GlcNAc binds between the AB and CD loops at the top of each of the three monomers of the MAdV-2 fibre head. However, infection competition assays show that soluble GlcNAc monosaccharide and natural GlcNAc-containing polymers do not inhibit infection by MAdV-2. Furthermore, site-directed mutation of the GlcNAc-binding residues does not prevent the inhibition of infection by soluble fibre protein. On the other hand, we show that the MAdV-2 fibre protein binds GlcNAc-containing mucin glycans, which suggests that the MAdV-2 fibre protein may play a role in viral mucin penetration in the mouse gut.

Avenues to Characterize the Interactions of Extended N-Glycans with Proteins by NMR Spectroscopy: The Influenza Hemagglutinin Case.

Long-chain multiantenna N-glycans are extremely complex molecules. Their inherent flexibility and the presence of repetitions of monosaccharide units in similar chemical environments hamper their full characterization by X-ray diffraction or standard NMR methods. Herein, the successful conformational and interaction analysis of a sialylated tetradecasaccharide N-glycan presenting two LacNAc repetitions at each arm is presented. This glycan has been identified as the receptor of the hemagglutinin protein of pathogenic influenza viruses. To accomplish this study, a N-glycan conjugated with a lanthanide binding tag has been synthesized, enabling analysis of the system by paramagnetic NMR. Under paramagnetic conditions, the NMR signals of each sugar unit in the glycan have been determined. Furthermore, a detailed binding epitope of the tetradecasaccharide N-glycan in the presence of HK/68 hemagglutinin is described.

Fluoroacetamide Moieties as NMR Spectroscopy Probes for the Molecular Recognition of GlcNAc-Containing Sugars: Modulation of the CH-π Stacking Interactions by Different Fluorination Patterns.

We herein propose the use of fluoroacetamide and difluoroacetamide moieties as sensitive tags for the detection of sugar-protein interactions by simple 1 H and/or 19 F NMR spectroscopy methods. In this process, we have chosen the binding of N,N'-diacetyl chitobiose, a ubiquitous disaccharide fragment in glycoproteins, by wheat-germ agglutinin (WGA), a model lectin. By using saturation-transfer difference (STD)-NMR spectroscopy, we experimentally demonstrate that, under solution conditions, the molecule that contained the CHF2 CONH- moiety is the stronger aromatic binder, followed by the analogue with the CH2 FCONH- group and the natural molecule (with the CH3 CONH- fragment). In contrast, the molecule with the CF3 CONH- isoster displayed the weakest intermolecular interaction (one order of magnitude weaker). Because sugar-aromatic CH-π interactions are at the origin of these observations, these results further contribute to the characterization and exploration of these forces and offer an opportunity to use them to unravel complex recognition processes.

Novel vaccines targeting dendritic cells by coupling allergoids to nonoxidized mannan enhance allergen uptake and induce functional regulatory T cells through programmed death ligand 1.

BACKGROUND: Allergen immunotherapy (AIT) is the only curative treatment for allergy. AIT faces pitfalls related to efficacy, security, duration, and patient compliance. Novel vaccines overcoming such inconveniences are in demand. OBJECTIVES: We sought to study the immunologic mechanisms of action for novel vaccines targeting dendritic cells (DCs) generated by coupling glutaraldehyde-polymerized grass pollen allergoids to nonoxidized mannan (PM) compared with glutaraldehyde-polymerized allergoids (P) or native grass pollen extracts (N). METHODS: Skin prick tests and basophil activation tests with N, P, or PM were performed in patients with grass pollen allergy. IgE-blocking experiments, flow cytometry, confocal microscopy, cocultures, suppression assays, real-time quantitative PCR, ELISAs, and ELISpot assays were performed to assess allergen capture by human DCs and T-cell responses. BALB/c mice were immunized with PM, N, or P. Antibody levels, cytokine production by splenocytes, and splenic forkhead box P3 (FOXP3)(+) regulatory T (Treg) cells were quantified. Experiments with oxidized PM were also performed. RESULTS: PM displays in vivo hypoallergenicity, induces potent blocking antibodies, and is captured by human DCs much more efficiently than N or P by mechanisms depending on mannose receptor- and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin-mediated internalization. PM endorses human DCs to generate functional FOXP3(+) Treg cells through programmed death ligand 1. Immunization of mice with PM induces a shift to nonallergic responses and increases the frequency of splenic FOXP3(+) Treg cells. Mild oxidation impairs these effects in human subjects and mice, demonstrating the essential role of preserving the carbohydrate structure of mannan. CONCLUSIONS: Allergoids conjugated to nonoxidized mannan represent suitable vaccines for AIT. Our findings might also be of the utmost relevance to development of therapeutic interventions in other immune tolerance-related diseases.

Breaking the Limits in Analyzing Carbohydrate Recognition by NMR Spectroscopy: Resolving Branch-Selective Interaction of a Tetra-Antennary N-Glycan with Lectins.

The biological recognition of complex-type N-glycans is part of many key physiological and pathological events. Despite their importance, the structural characterization of these events remains unsolved. The inherent flexibility of N-glycans hampers crystallization and the chemical equivalence of individual branches precludes their NMR characterization. By using a chemoenzymatically synthesized tetra-antennary N-glycan conjugated to a lanthanide binding tag, the NMR signals under paramagnetic conditions discriminated all four N-acetyl lactosamine antennae with unprecedented resolution. The NMR data revealed the conformation of the N-glycan and permitted for the first time the direct identification of individual branches involved in the recognition by two N-acetyllactosamine-binding lectins, Datura stramonium seed lectin (DSL) and Ricinus Communis agglutinin (RCA120).

Intra- and intermolecular interactions of human galectin-3: assessment by full-assignment-based NMR.

Galectin-3 is an adhesion/growth-regulatory protein with a modular design comprising an N-terminal tail (NT, residues 1-111) and the conserved carbohydrate recognition domain (CRD, residues 112-250). The chimera-type galectin interacts with both glycan and peptide motifs. Complete (13)C/(15)N-assignment of the human protein makes NMR-based analysis of its structure beyond the CRD possible. Using two synthetic NT polypeptides covering residues 1-50 and 51-107, evidence for transient secondary structure was found with helical conformation from residues 5 to 15 as well as proline-mediated, multi-turn structure from residues 18 to 32 and around PGAYP repeats. Intramolecular interactions occur between the CRD F-face (the 5-stranded ß-sheet behind the canonical carbohydrate-binding 6-stranded ß-sheet of the S-face) and NT in full-length galectin-3, with the sequence P(23)GAW(26)…P(37)GASYPGAY(45) defining the primary binding epitope within the NT. Work with designed peptides indicates that the PGAX motif is crucial for self-interactions between NT/CRD. Phosphorylation at position Ser6 (and Ser12) (a physiological modification) and the influence of ligand binding have minimal effect on this interaction. Finally, galectin-3 molecules can interact weakly with each other via the F-faces of their CRDs, an interaction that appears to be assisted by their NTs. Overall, our results add insight to defining binding sites on galectin-3 beyond the canonical contact area for ß-galactosides.

Detailed Investigation of the Immunodominant Role of O-Antigen Stoichiometric O-Acetylation as Revealed by Chemical Synthesis, Immunochemistry, Solution Conformation and STD-NMR Spectroscopy for Shigella flexneri†3a.

Shigella flexneri 3a causes bacillary dysentery. Its O-antigen has the {2)-[α-d-Glcp-(1→3)]-α-l-Rhap-(1→2)-α-l-Rhap-(1→3)-[Ac→2]-α-l-Rhap-(1→3)-[Ac→6]≈40 % -ß-d-GlcpNAc-(1→} ([(E)ABAc CAc D]) repeating unit, and the non-O-acetylated equivalent defines S. flexneri X. Propyl hepta-, octa-, and decasaccharides sharing the (E')A'BAc CD(E)A sequence, and their non-O-acetylated analogues were synthesized from a fully protected BAc CD(E)A allyl glycoside. The stepwise introduction of orthogonally protected mono- and disaccharide imidate donors was followed by a two-step deprotection process. Monoclonal antibody binding to twenty-six S. flexneri types 3a and X di- to decasaccharides was studied by an inhibition enzyme-linked immunosorbent assay (ELISA) and STD-NMR spectroscopy. Epitope mapping revealed that the 2C -acetate dominated the recognition by monoclonal IgG and IgM antibodies and that the BAc CD segment was essential for binding. The glucosyl side chain contributed to a lesser extent, albeit increasingly with the chain length. Moreover, tr-NOESY analysis also showed interaction but did not reveal any meaningful conformational change upon antibody binding.

The Quest for Anticancer Vaccines: Deciphering the Fine-Epitope Specificity of Cancer-Related Monoclonal Antibodies by Combining Microarray Screening and Saturation Transfer Difference NMR.

The identification of MUC1 tumor-associated Tn antigen (αGalpNAc1-O-Ser/Thr) has boosted the development of anticancer vaccines. Combining microarrays and saturation transfer difference NMR, we have characterized the fine-epitope mapping of a MUC1 chemical library (naked and Tn-glycosylated) toward two families of cancer-related monoclonal antibodies (anti-MUC1 and anti-Tn mAbs). Anti-MUC1 mAbs clone VU-3C6 and VU-11E2 recognize naked MUC1-derived peptides and bind GalNAc in a peptide-sequence-dependent manner. In contrast, anti-Tn mAbs clone 8D4 and 14D6 mostly recognize the GalNAc and do not bind naked MUC1-derived peptides. These anti-Tn mAbs show a clear preference for glycopeptides containing the Tn-Ser antigen rather than the Tn-Thr analogue, stressing the role of the underlying amino acid (serine or threonine) in the binding process. The reported strategy can be employed, in general, to unveil the key minimal structural features that modulate antigen-antibody recognition, with particular relevance for the development of Tn-MUC1-based anticancer vaccines.

A murine monoclonal antibody to glycogen: characterization of epitope-fine specificity by saturation transfer difference (STD) NMR spectroscopy and its use in mycobacterial capsular α-glucan research.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a major pathogen responsible for 1.5 million deaths annually. This bacterium is characterized by a highly unusual and impermeable cell envelope, which plays a key role in mycobacterial survival and virulence. Although many studies have focused on the composition and functioning of the mycobacterial cell envelope, the capsular α-glucan has received relatively minor attention. Here we show that a murine monoclonal antibody (Mab) directed against glycogen cross-reacts with mycobacterial α-glucans, polymers of α(1-4)-linked glucose residues with α(1-6)-branch points. We identified the Mab epitope specificity by saturation transfer difference NMR and show that the α(1-4)-linked glucose residues are important in glucan-Mab interaction. The minimal epitope is formed by (linear) maltotriose. Notably, a Mycobacterium mutant lacking the branching enzyme GlgB does not react with the Mab; this suggests that the α(1-6)-branches form part of the epitope. These seemingly conflicting data can be explained by the fact that in the mutant the linear form of the α-glucan (amylose) is insoluble. This Mab was subsequently used to develop several techniques helpful in capsular α-glucan research. By using a capsular glucan-screening methodology based on this Mab we were able to identify several unknown genes involved in capsular α-glucan biogenesis. Additionally, we developed two methods for the detection of capsular α-glucan levels. This study therefore opens new ways to study capsular α-glucan and to identify possible targets for further research.

Recent Developments in Synthetic Carbohydrate-Based Diagnostics, Vaccines, and Therapeutics.

Glycans are everywhere in biological systems, being involved in many cellular events with important implications for medical purposes. Building upon a detailed understanding of the functional roles of carbohydrates in molecular recognition processes and disease states, glycans are increasingly being considered as key players in pharmacological research. On the basis of the important progress recently made in glycochemistry, glycobiology, and glycomedicine, we provide a complete overview of successful applications and future perspectives of carbohydrates in the biopharmaceutical and medical fields. This review highlights the development of carbohydrate-based diagnostics, exemplified by glycan imaging techniques and microarray platforms, synthetic oligosaccharide vaccines against infectious diseases (e.g., HIV) and cancer, and finally carbohydrate-derived therapeutics, including glycomimetic drugs and glycoproteins.

Monitoring Glycan-Protein Interactions by NMR Spectroscopic Analysis: A Simple Chemical Tag That Mimics Natural CH-π Interactions.

Detection of molecular recognition processes requires robust, specific, and easily implementable sensing methods, especially for screening applications. Here, we propose the difluoroacetamide moiety (an acetamide bioisoster) as a novel tag for detecting by NMR analysis those glycan-protein interactions that involve N-acetylated sugars. Although difluoroacetamide has been used previously as a substituent in medicinal chemistry, here we employ it as a specific sensor to monitor interactions between GlcNAc-containing glycans and a model lectin (wheat germ agglutinin). In contrast to the widely employed trifluoroacetamide group, the difluoroacetamide tag contains geminal (1) H and (19) F atoms that allow both (1) H and (19) F NMR methods for easy and robust detection of molecular recognition processes involving GlcNAc- (or GalNAc-) moieties over a range of binding affinities. The CHF2 CONH- moiety behaves in a manner that is very similar to that of the natural acetamide fragment in the involved aromatic-sugar interactions, providing analogous binding energy and conformations, whereas the perfluorinated CF3 CONH- analogue differs more significantly.

Conformational Plasticity in Glycomimetics: Fluorocarbamethyl-L-idopyranosides Mimic the Intrinsic Dynamic Behaviour of Natural Idose Rings.

Sugar function, structure and dynamics are intricately correlated. Ring flexibility is intrinsically related to biological activity; actually plasticity in L-iduronic rings modulates their interactions with biological receptors. However, the access to the experimental values of the energy barriers and free-energy difference for conformer interconversion in water solution has been elusive. Here, a new generation of fluorine-containing glycomimetics is presented. We have applied a combination of organic synthesis, NMR spectroscopy and computational methods to investigate the conformational behaviour of idose- and glucose-like rings. We have used low-temperature NMR spectroscopic experiments to slow down the conformational exchange of the idose-like rings. Under these conditions, the exchange rate becomes slow in the (19) F NMR spectroscopic chemical shift timescale and allows shedding light on the thermodynamic and kinetic features of the equilibrium. Despite the minimal structural differences between these compounds, a remarkable difference in their dynamic behaviour indeed occurs. The importance of introducing fluorine atoms in these sugars mimics is also highlighted. Only the use of (19) F NMR spectroscopic experiments has permitted the unveiling of key features of the conformational equilibrium that would have otherwise remained unobserved.

Lanthanide-chelating carbohydrate conjugates are useful tools to characterize carbohydrate conformation in solution and sensitive sensors to detect carbohydrate-protein interactions.

The increasing interest in the functional versatility of glycan epitopes in cellular glycoconjugates calls for developing sensitive methods to define carbohydrate conformation in solution and to characterize protein-carbohydrate interactions. Measurements of pseudocontact shifts in the presence of a paramagnetic cation can provide such information. In this work, the energetically privileged conformation of a disaccharide (lactose as test case) was experimentally inferred by using a synthetic carbohydrate conjugate bearing a lanthanide binding tag. In addition, the binding of lactose to a biomedically relevant receptor (the human adhesion/growth-regulatory lectin galectin-3) and its consequences in structural terms were defined, using Dy(3+), Tb(3+), and Tm(3+). The described approach, complementing the previously tested protein tagging as a way to exploit paramagnetism, enables to detect binding, even weak interactions, and to characterize in detail topological aspects useful for physiological ligands and mimetics in drug design.

Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner.

Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with (15)N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.

Systematic dissection of an aminopyrrolic cage receptor for ß-glucopyranosides reveals the essentials for effective recognition.

A set of structures designed for the recognition of glucosides has been obtained by systematically destructuring a tripodal aminopyrrolic cage receptor that selectively recognizes octyl-ß-D-glucopyranoside (OctßGlc). NMR spectroscopy and isothermal titration calorimetry binding measurements showed that cleavage of one pillar of the cage was beneficial to the binding properties of the receptor, as long as two residual amino groups of the cleaved pillar were present. Removal of these two residual amino groups produced a dramatic loss of affinity for OctßGlc of the resulting monocyclic analogue of the parent cage receptor. A significant improvement in the binding ability was achieved by replacing one pillar with two aminopyrrolic hydrogen-bonding arms, despite the loss of a preorganized structure. In contrast to the cage receptor, recognition of OctßGlc was observed, even in a competitive medium (30 % DMF in chloroform). Structural studies in solution, carried out through NMR spectroscopy and molecular modeling calculations, led to the elucidation of the 3D binding modes of the side-armed monocyclic receptors; this highlighted the key role of the amino groups and demonstrated the occurrence of a rotaxane-like complex, which featured the octyl chain of the glucoside threaded through the macrocyclic ring.

Delineating binding modes of Gal/GalNAc and structural elements of the molecular recognition of tumor-associated mucin glycopeptides by the human macrophage galactose-type lectin.

The human macrophage galactose-type lectin (MGL) is a key physiological receptor for the carcinoma-associated Tn antigen (GalNAc-α-1-O-Ser/Thr) in mucins. NMR and modeling-based data on the molecular recognition features of synthetic Tn-bearing glycopeptides by MGL are presented. Cognate epitopes on the sugar and matching key amino acids involved in the interaction were identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non-glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for MGL binding, as is Ca(2+) . NMR data were complemented with molecular dynamics simulations and Corcema-ST to establish a 3D view on the molecular recognition process between Gal, GalNAc, and the Tn-presenting glycopeptides and MGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH-π contacts involving exclusively the NHAc moiety.

Lactose binding to human galectin-7 (p53-induced gene 1) induces long-range effects through the protein resulting in increased dimer stability and evidence for positive cooperativity.

The product of p53-induced gene 1 is a member of the galectin family, i.e., galectin-7 (Gal-7). To move beyond structural data by X-ray diffraction, we initiated the study of the lectin by nuclear magnetic resonance (NMR) and circular dichroism spectroscopies, and molecular dynamics (MD) simulations. In concert, our results indicate that lactose binding to human Gal-7 induces long-range effects (minor conformational shifts and changes in structural dynamics) throughout the protein that result in stabilization of the dimer state, with evidence for positive cooperativity. Monte Carlo fits of (15)N-Gal-7 HSQC titrations with lactose using a two-site model yield K1 = 0.9 ± 0.6 × 10(3) M(-1) and K2 = 3.4 ± 0.8 × 10(3) M(-1). Ligand binding-induced stabilization of the Gal-7 dimer was supported by several lines of evidence: MD-based calculations of interaction energies between ligand-loaded and ligand-free states, gel filtration data and hetero-FRET spectroscopy that indicate a highly reduced tendency for dimer dissociation in the presence of lactose, CD-based thermal denaturation showing that the transition temperature of the lectin is significantly increased in the presence of lactose, and saturation transfer difference (STD) NMR using a molecular probe of the monomer state whose presence is diminished in the presence of lactose. MD simulations with the half-loaded ligand-bound state also provided insight into how allosteric signaling may occur. Overall, our results reveal long-range effects on Gal-7 structure and dynamics, which factor into entropic contributions to ligand binding and allow further comparisons with other members of the galectin family.